Esmée Ruizendaal

Background: Cryptosporidium species and Giardia lamblia are endemic gastrointestinal pathogens worldwide, whereas Entamoeba histolytica is less commonly endemic, but can cause invasive disease. Due to limited surveillance of these parasites, epidemiological data in the Netherlands are scarce. Hence, we conducted a retrospective analysis of Nucleic Acid Amplification Test (NAAT) results to assess the incidence, disease burden, seasonality, and epidemiological trends of these infections.Methods: This retrospective, nationally representative cohort study analysed NAAT results from 24 medical microbiology laboratories (MMLs) (2015–2024) to estimate the incidence of Cryptosporidium species, G. lamblia, and E. histolytica, and to quantify the disease burden of human cryptosporidiosis and giardiasis in disability-adjusted life years (DALY) in the Netherlands. The study included all individuals tested for these parasites at participating MMLs. National incidence was estimated by extrapolating the population covered by MMLs, using NAAT-to-population ratios from six laboratories with known catchment sizes.Findings: A total of 9,373 Cryptosporidium species, 20,898 G. lamblia and 417 E. histolytica cases were identified, leading to an estimated mean incidence of 9.75 (95% CI: 9.56–9.95), 21.80 (21.40–22.00) and 0.43 (0.39–0.48) per 100,000 population, respectively. The annual disease burden (undiscounted DALYs) ranged from 74 (95% CI: 28-143) to 378 (95%CI: 150-694) for cryptosporidiosis (2020–2023), and from 233 (131–442) to 500 (275–950) for giardiasis (2021–2019). Cryptosporidium species and G. lamblia infections peaked in summer/fall and were most common in children, while E. histolytica incidence was low, non-seasonal, and significantly higher in men, affecting both children and adults.Interpretation: The persistently high disease burden of giardiasis and the recent, strong seasonal rise in cryptosporidiosis, highlight the need for enhanced surveillance, including genotyping and travel history, to guide public health interventions. Although E. histolytica incidence is low, its potential severity may warrant targeted measures such as contact tracing.

Diagnosis of malaria in pregnancy is problematic due to the low sensitivity of conventional diagnostic tests (rapid diagnostic test and microscopy), which is exacerbated due to low peripheral parasite densities, and lack of clinical symptoms. In this study, six potential biomarkers to support malaria diagnosis in pregnancy were evaluated. Blood samples were collected from pregnant women at antenatal clinic visits and at delivery. Microscopy and real-time PCR were performed for malaria diagnosis and biomarker analyses were performed by ELISA (interleukin 10, IL-10; tumor necrosis factor-α, TNF-α; soluble tumor necrosis factor receptor II, sTNF-RII; soluble fms-like tyrosine kinase 1, sFlt-1; leptin and apolipoprotein B, Apo-B). A placental biopsy was collected at delivery to determine placental malaria. IL-10 and sTNF-RII were significantly higher at all time-points in malaria-infected women (p < 0.001). Both markers were also positively associated with parasite density (p < 0.001 and p = 0.003 for IL-10 and sTNF-RII respectively). IL-10 levels at delivery, but not during pregnancy, were negatively associated with birth weight. A prediction model was created using IL-10 and sTNF-RII cut-off points. For primigravidae the model had a sensitivity of 88.9% (95%CI 45.7–98.7%) and specificity of 83.3% (95% CI 57.1–94.9%) for diagnosing malaria during pregnancy. For secundi- and multigravidae the sensitivity (81.8% and 56.5% respectively) was lower, while specificity (100.0% and 94.3% respectively) was relatively high. Sub-microscopic infections were detected in 2 out of 3 secundi- and 5 out of 12 multigravidae. The combination of biomarkers IL-10 and sTNF-RII have the potential to support malaria diagnosis in pregnancy. Additional markers may be needed to increase sensitivity and specificity, this is of particular importance in populations with sub-microscopic infections or in whom other inflammatory diseases are prevalent.

One of the current strategies to prevent malaria in pregnancy is intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP). However, in order for pregnant women to receive an adequate number of SP doses, they should attend a health facility on a regular basis. In addition, SP resistance may decrease IPTp-SP efficacy. New or additional interventions for preventing malaria during pregnancy are therefore warranted. Because it is known that community health workers (CHWs) can diagnose and treat malaria in children, in this study screening and treatment of malaria in pregnancy by CHWs was evaluated as an addition to the regular IPTp-SP program. CHWs used rapid diagnostic tests (RDTs) for screening and artemether–lumefantrine was given in case of a positive RDT. Overall, CHWs were able to conduct RDTs with a sensitivity of 81.5% (95% confidence interval [CI] 67.9–90.2) and high specificity of 92.1% (95% CI 89.9–93.9) compared with microscopy. After a positive RDT, 79.1% of women received artemether–lumefantrine. When treatment was not given, this was largely due to the woman being already under treatment. Almost all treated women finished the full course of artemether–lumefantrine (96.4%). In conclusion, CHWs are capable of performing RDTs with high specificity and acceptable sensitivity, the latter being dependent on the limit of detection of RDTs. Furthermore, CHWs showed excellent adherence to test results and treatment guidelines, suggesting they can be deployed for screen and treat approaches of malaria in pregnancy.

Pregnant women are a high-risk group for Plasmodium falciparum infections, which may result in maternal anaemia and low birth weight newborns, among other adverse birth outcomes. Intermittent preventive treatment with sulfadoxine–pyrimethamine during pregnancy (IPTp-SP) is widely implemented to prevent these negative effects of malaria. However, resistance against SP by P. falciparum may decrease efficacy of IPTp-SP. Combinations of point mutations in the dhps (codons A437, K540) and dhfr genes (codons N51, C59, S108) of P. falciparum are associated with SP resistance. In this study the prevalence of SP resistance mutations was determined among P. falciparum found in pregnant women and the general population (GP) from Nanoro, Burkina Faso and the association of IPTp-SP dosing and other variables with mutations was studied. Blood spots on filter papers were collected from pregnant women at their first antenatal care visit (ANC booking) and at delivery, from an ongoing trial and from the GP in a cross-sectional survey. The dhps and dhfr genes were amplified by nested PCR and products were sequenced to identify mutations conferring resistance (ANC booking, n = 400; delivery, n = 223; GP, n = 400). Prevalence was estimated with generalized estimating equations and for multivariate analyses mixed effects logistic regression was used. The prevalence of the triple dhfr mutation was high, and significantly higher in the GP and at delivery than at ANC booking, but it did not affect birth weight. Furthermore, quintuple mutations (triple dhfr and double dhps mutations) were found for the first time in Burkina Faso. IPTp-SP did not significantly affect the occurrence of any of the mutations, but high transmission season was associated with increased mutation prevalence in delivery samples. It is unclear why the prevalence of mutations was higher in the GP than in pregnant women at ANC booking. The high number of mutants and the presence of quintuple mutants in Burkina Faso confirm concerns about the efficacy of IPTp-SP in the near future. Other drug combinations to tackle malaria in pregnancy should, therefore, be explored. An increase in mutation prevalence due to IPTp-SP dosing could not be confirmed.